Tissue engineering in the nervous system deals with the functional replacement of damaged tissues and nervous system regeneration.
The ability to organize cells in three dimensions (3-D) is an important component of tissue engineering. The behavior of cells is influenced both by their intrinsic genetic programs and their extracellular environment. The extracellular environment includes `passive` structural components and biologically `active` components.
Most cells in multicellular organisms are in contact with an intricate meshwork of interacting, extracellular macromolecules that constitute the extracellular matrix (ECM). These macromolecules, mainly proteins and polysaccharides, are secreted locally and assemble into an organized 3-D meshwork in the extracellular spaces of most tissues. ECM molecules include glycosaminoglycans, and proteoglycans such as chrondroitin sulfate, fibronectin, heparin sulfate, hyaluron dermatan sulfate, keratin sulfate, laminin, collagen, heparan sulfate proteoglycan, and elastin. In addition to serving as a universal biological glue, ECM molecules also form highly specialized structures such as cartilage, tendons, basal laminae, and (in conjunction with secondary deposition of calcium phosphate) bone and teeth. Alberts et al., Molecular Biology of the Cell, Garland, N.Y., pp. 802-24 (1989).
Extracellular matrices modulate the organization of the intracellular cytoskeleton, cell differentiation and the spatial architecture of cells and tissues. In act, the ECM plays a critical role in regulating the behaviour of cells that contact it by influencing cellular development, migration, proliferation, differentiation, shape, polarity and metabolic function.
Several peptide active sites responsible for cell attachment have been identified in various ECM molecules.
In vivo laminin (LN) immunoreactivity has been detected in several regions of the embryo including muscles (Chui and Sanes, Dev. Biol., 103, pp. 456-67 (1984)), spinal cord (Azzi et al., Matrix, 9, pp. 479-85 (1989), spinal roots (Rogers et al., Dev. Biol., 113, pp. 429-35 (1986)), optic nerve (McLoon et al., J. Neurosci., 8, pp. 1981-90 (1988)), cerebral cortex (Liesi, EMBO, 4, pp. 1163-70 (1985); Zhou, Dev. Brain Res., 55, pp. 191-201 (1990)), hippocampus (Gordon-Weeks et al., J. Neurocytol., 18, pp. 451-63 (1989)) and the medial longitudinal fasciculus of the midbrain (Letourneau et al., Development, 105, pp. 505-19 (1989)).
The tripeptidic sequence RGD (ArgGlyAsp; AA.sub.2 -AA.sub.4 of SEQ ID NO:2) has been identified to be responsible for some of the cell adhesion properties of fibronectin (Pierschbacher and Ruoslahti, Science, 309, pp. 30-33 (1984)), laminin (Grant et al., Cell, 58, pp. 933-43 (1989)), entactin (Durkin et al., J. Cell. Biol., 107, pp. 2329-40 (1988)), vitronectin (Suzuki et al., EMBO, 4, pp. 2519-24 (1985)), collagen I (Dedhar et al., J. Cell. Biol., 107, pp. 2749-56 (1987)), collagen IV (Aumailley et al., Exp. Cell Res., 187, pp. 463-74 (1989)), thrombospondin (Lawler et al., J. Cell. Biol., 107, pp. 2351-61 (1988)) and tenascin (Friedlander et al., J. Cell. Biol., 107, pp. 2329-40 (1988)).
The sequence YIGSR (TyrIleGlySerArg; AA.sub.5 -AA.sub.9 of SEQ ID NO:1), found on the B1 chain of laminin, promotes epithelial cell attachment (Graf et al., Biochemistry, 26, pp. 6896-900 (1987)) and inhibits tumor metastasis (Iwamoto et al., Science, 238, pp. 1132-34 (1987)).
The IKVAV sequence found on the A chain of laminin, has been reported to promote neurite outgrowth (Tashiro et al., J. Biol. Chem., 264, pp. 16174-182 (1989); Jucker et al., J. Neurosci. Res. 28, pp. 507-17 (1991)).
All of the studies using these preptidic sequences of cell attachment and neurite promotion were conducted on flat two-dimension substrates (Smallheiser et al., Dev. Brain Res., 12, pp. 136-40 (1984); Graf et al., Biochemistry, 26, pp. 6896-900 (1987); Sephel et al., Biochem. Biophys. Res. Comm., 2, pp. 821-29 (1989); Jucker et al., J. Neurosci. Res., 28, pp. 507-17 (1991)). The physical and chemical nature of the culture substrate influences cell attachment and neurite extension. The physical microstructure of a 2-D culture substrate can influence cell behavior. The use of permissive and on-permissive culture surface chemistries facilitates nerve guidance in 2-D. The cell attachment regulating function of various serum proteins like albumin and fibronectin is dependent on the chemistries of the culture substrates that they are adsorbed onto.
Gene expression is reported to be regulated differently by a flat 2-D substrate as opposed to a hydrated 3-D substrate. For example, monolayer culture of primary rabbit articular chondrocyte and human epiphyseal chondrocyte on 2-D tissue culture substrates causes primary chondrocyte to lose their differentiated phenotype. The differentiated chondrocyte phenotype is re-expressed when they are cultured in 3-D agarose gels (Benya and Shaffer, Cell, 30, pp. 215-24 (1982); Aulthouse, et al., In Vitro Cell Dev. Bio., 25, pp. 659-68 (1989)).
Similarly, alkaline phosphatase gene expression in primary bile ductular epithelial cells is differentially regulated when they are cultured in 3-D Matrigel.RTM., collagen or agarose gels are opposed to 2-D cultures (Mathis et al., Cancer Res., 48, pp. 6145-53 (1988)).
Therefore, 3-D presentation of ECM components may better mimic the in vivo environment in influencing cell or tissue response. In particular, in vivo use of ECM biomolecules may require such a 3-D system for optimal efficacy. The development of a defined, bioartificial 3-D matrix that presents ECM molecules, or active portions thereof, would facilitate tissue engineering in the nervous system by allowing in vitro and in vivo cell manipulation and cell culture in 3-D. See, e.g., Koebe et al., Cryobiology, 27, pp. 576-84 (1990).
In addition, there is a need to develop a defined, biosynthetic matrix, because the tumorogenic origins of some commercially available ECM, e.g., Matrigel.RTM. mouse sarcoma derived ECM, render it unattractive for some in vitro and in vivo applications. Further, naturally occuring ECM components such as collagen may be enzymatically degraded in the body while a synthetic ECM is less likely to be degraded.